Phosphorylation is the most frequently occurring reversible post-translational modification of proteins. The regulation of phosphorylation and the effects of phosphorylation are exceedingly complex. One means of tackling the complexity is to study the properties of individual protein kinases. This proposal focuses on two major questions regarding a ubiquitous protein-serine kinase, known as casein kinase I (CKI), whose physiological roles remain unknown. The first is the regulation of the enzyme and the second is the function of the enzyme. Thus, the objectives of these experiments are: 1) to demonstrate that CKI is the insulin-activated casein kinase detected in high speed particulate fractions of insulin-treated cells and in livers from insulin-treated rabbits; 2) to discover the molecular events underlying the regulation of CKI by insulin; 3) to measure the effect on substrate function of physiologically relevant phosphorylations catalyzed by CKI and, if necessary, to determine consensus sequences for CKI in order to synthesize a peptide substrate to use in studies of its regulation in cells; 4) to study the physical basis for the association of CKI with membranes; and 5) to clone CKI to gain insight into its physiological roles both by examining its distribution and expression in mammals and by identifying homologs in yeast and/or Drosophila so that in the future genetic techniques may be used to probe its function.